Laser scanning confocal fluorescence microscopy was used to assess cell viability in allograft heart valves. Paraffin sections of formalin-fixed valve tissues were stained for the simultaneous demonstration of DNA (DAPI; blue fluorescence), apoptosis (incorporation of fluorescein-labeled deoxynucleotides by terminal deoxynucleotidyl transferase; green fluorescence) and immunostaining for either Factor VIII for endothelium or proliferating cell nuclear antigen (PCNA) (Texas red-labeled antibodies; red fluorescence). Native ovine aortic valves (n=4), explanted ovine allografts (n=8) (implanted as thoracic aortic valved conduits in sheep) and a human allograft valve explanted because of calcification 5 years and 9 months after implantation for pulmonary atresia were evaluated. Results obtained indicated that: 1) a marked reduction in mitotic potential of leaflet cells occurs within two days of implantation; 2) endothelial cells are progressively lost after implantation, and 3) apoptosis occurs after two days of implantation in connective tissue cells, and leads to the formation of numerous apoptotic bodies and progressive decrease in leaflet cellularity. By 20 weeks after implantation, the allograft valve leaflets were essentially acellular with the exception of rare myofibroblasts and a moderate number of macrophages (thought to be of host origin); no intra or extracellular apoptotic bodies and no PCNA-positive cells were observed at this time. However, at this time the cusps had undergone marked thickening and contraction due to the formation of extensive fibrous sheathing by host tissue. Similar findings were present in the human allograft. Thus, apoptosis is a major cause of loss of cell viability in implanted allografts. Further application of the methods used in this study should increase our understanding of these changes.